Technical Field
The present invention relates to a highly stable nucleic acid-polysaccharide complex of an siRNA and schizophyllan. Also, the present invention relates to a method for controlling the function of a Dectin-1 expressing cell such as a dendritic cell and obtaining an effective therapeutic effect by inhibiting expression of a target gene using the nucleic acid-polysaccharide complex, and to a drug for use in the method. Moreover, the present invention relates to a method for immunomodulation and a drug for use therein. In particular, the present invention relates to a drug or the like for use in the suppression of rejection occurring in organ transplantation and for use in the treatment of allergies and autoimmune diseases, using an siRNA against a costimulatory factor and a highly stable nucleic acid-polysaccharide complex of schizophyllan.
Background Art
RNA interference (RNAi) discovered in 1998 has markedly superior efficacy and persistence to conventional antisense methods and is a breakthrough gene expression inhibitory method, and therefore there has been a hope for its pharmaceutical applications. However, a double strand RNA (i.e., siRNA) that shows an RNAi activity is often decomposed during the process from administration to uptake into a target cell or decomposed in a cell, and it has been difficult to form a RISC complex, i.e., its active substance, in a cell. Accordingly, even though it is a superior gene expression inhibitory method, a sufficient effect is not obtainable, and therefore a pharmaceutical product that uses an siRNA is not yet available.
Unmodified siRNAs are decomposed by nuclease that is present in, for example, blood, and few unmodified siRNAs demonstrate an RNAi effect in a target cell. Accordingly, various chemical modifications to give nuclease resistance have been performed on siRNAs. Nevertheless, a high dosage is necessary for effective introduction into a cell. Also, it is known that because administration of a double strand nucleotide into a living body in a high dosage enhances a natural immunoreaction, an unintended effect, i.e., immunostimulatory reaction, appears. Accordingly, a delivery technique to specifically introduce an siRNA into a target cell is necessary. For siRNA delivery techniques, techniques to embed an siRNA, such as liposomes and macromolecular nanomicelles, have been developed. However, in terms of target tropism, these delivery techniques are still within the meaning of passive targeting, and in order to overcome this shortcoming, a procedure for imparting, for example, a molecule that binds to the target cell to an siRNA drug is needed.
With such conventional art as background, a demand exists for positive targeting and a delivery technique for an siRNA that shows an RNAi activity significantly within the target cell. Accordingly, as a delivery method of an siRNA to a dendritic cell, a complex of schizophyllan and a polydeoxyadenine-added siRNA has been proposed (see WO 2009/078470). However, it is not necessarily possible to obtain a satisfactory therapeutic effect with the technique of WO 2009/078470.
On the other hand, it has been reported so far that phosphorothioated polydeoxyadenine can form a stable complex with schizophyllan (see Bull. Chem. Soc. Jpn., 77, 1101-1110 (2004)). However, it is thought that a complex of schizophyllan and an siRNA to which phosphorothioated polydeoxyadenine is added is not possible to exhibit an effective RNA interference effect for the following reasons. In the complex of schizophyllan and a polydeoxyadenine-added siRNA, the complexing portion between polydeoxyadenine and schizophyllan serves as a steric hindrance in the formation of a RISC complex. Therefore, in the complex of schizophyllan and a polydeoxyadenine-added siRNA, the portion where the complex with schizophyllan is formed needs to be removed before forming a RISC complex in a cell, but phosphorothioated polydeoxyadenine and schizophyllan have markedly high stability, thus being not easily removable. In addition, because phosphorothioated polydeoxyadenine is unlikely to be enzymatically resolved, it is difficult to remove the portion where a complex is formed between schizophyllan and an siRNA to which phosphorothioated polydeoxyadenine is added (in particular, a 21 mer type siRNA that is not affected by the action of Dicer). Therefore, it is thought that with regard to the complex of schizophyllan and an siRNA to which phosphorothioated polydeoxyadenine is added (in particular, a 21 mer type siRNA), it is not possible to separate the phosphorothioated polydeoxyadenine and schizophyllan from the siRNA in a cell, thus making it difficult to form a RISC complex.
Therefore, currently, it is thought that a complex of schizophyllan and an siRNA to which phosphorothioated polydeoxyadenine is added (in particular, a 21 mer type siRNA) is not usable from the technical viewpoint of effectively exhibiting an RNA interference effect.